The long term goal of our proposed research is to understand at the molecular level how enzymes achieve their extraordinary catalytic efficiencies. We seek to do this through direct observation of enzyme- bound ligands (substrates, intermediates, and transition-state analogs). We will use techniques (NMR, IR Spectroscopies) sensitive to local bond distortions, electronic environment, protonation state and other factors likely to reflect the enzyme's catalytic strategies. We seek to do this through observations of the effects of perturbations (pH, temperature, structure of enzyme and ligand) on the standard free energies of enzyme- bound substrates, intermediates, transition-states and analogs of those states. We will use techniques (various spectroscopic titrations, calorimetry, transient kinetics) appropriate for the measurement of equilibrium and rate constants. We will make quantitative comparisons (linear free energy relationships) with the effects of these same perturbations on well-understood model systems leading to an understanding of the molecular basis of those interactions between substrate and enzyme which lead to efficient catalysis. We will focus on two groups of enzymes; the Claisen enzymes (citrate synthase) and the nucleoside aminohydrolases (adenosine deaminase). The Claisen enzymes are prominent in pathways requiring carbon-carbon bond formation in the biosynthesis of fats and cholesterol as well in the energy-yielding pathways of the tricarboxylic acid cycle and the glyoxylate cycle. These enzymes operate through activation of a substrate carbonyl together with the stabilization of an activated form of the acylthioester substrate. Enzymes which catalyze amino-nucleoside(tide) hydrolysis have more varied but no less vital roles. Adenosine deaminase is necessary to the integrity of the immune response. Other enzymes of this class participate in salvage pathways of purines and pyrimidines or are involved in the maintenance of cellular energy balance. These enzymes operate through stabilization of tetrahedral intermediates, a catalytic strategy used by several other important enzyme groups. The necessary groundwork is near completion and new results are rapidly accumulating. In previous work, we have identified catalytic strategies used by the two enzymes. We have developed probes to specifically detect and quantify the effects of perturbations on the catalytic apparatus; we have prepared enzymes with single- and multiple- site changes. We are now prepared to characterize these mutants and to continue our search for additional strategies in both groups. An understanding of how enzymes work is necessary to understand at the most fundamental level the metabolic processes in normal and disease states.